469883-75-8Relevant academic research and scientific papers
Labeling proteins with fluorophore/thioamide Foerster resonant energy transfer pairs by combining unnatural amino acid mutagenesis and native chemical ligation
Wissner, Rebecca F.,Batjargal, Solongo,Fadzen, Colin M.,Petersson, E. James
, p. 6529 - 6540 (2013)
We have recently shown that p-cyanophenylalanine (Cnf) and a thioamide can be used as a minimally perturbing Foerster resonant energy transfer (FRET) pair to monitor protein conformation. We have also shown that thioamide analogues of natural amino acids can be incorporated into full-sized proteins through native chemical ligation. For intermolecular studies with Cnf/thioamide FRET pairs, Cnf can be incorporated into proteins expressed in Escherichia coli through unnatural amino acid mutagenesis using a Cnf-specific tRNA synthetase. For intramolecular studies, a Cnf-labeled protein fragment can be expressed in E. coli and then ligated to a thioamide-labeled peptide synthesized on solid phase. This combination of methods allows for rapid access to double-labeled proteins with a minimum of unnecessary chemical synthesis. We demonstrate the utility of this approach by studying the binding of peptides to the protein calmodulin and by determining the orientation of the N- and C-termini in the amyloidogenic protein α-synuclein.
